William N. Fishbein

Quantitative densitometry of 1–50 μg protein in acrylamide gel slabs with coomassie blue☆

Abstract A critical study has been made of quantitative densitometry with Coomassie blue in acrylamide gel slabs, using successive applications of a pure protein, α-urease, to provide peaks well separated by reproducible baselines, a densitometer calibrated for linear response with optical density, and repetitive digital area integration. Staining was proportional to protein weight over a range that was markedly dependent upon migration distance in the gel. For bands that had migrated more than 9 cm, linearity was followed over a range of 1–55 μg. At shorter migration distances the range of linearity progressively contracted, until at 2 cm migration it encompassed only 1–12 μg. This pattern suggests that progressive compaction of the protein bands at shorter migration distances is the responsible factor, preventing the stoichiometric uptake of dye molecules once a critical protein concentration has been exceeded. Accepting a 10–15% error in densitometry, the deviations could be corrected by application of an empirically derived set of factors, so that a 1–50 μg range could be quantitated for any distance beyond 2 cm from the slot origin. The correction factors, although derived for 5% acrylamide gels, were also applicable to other gel strengths.

Natural gastric infection with Helicobacter pylori in monkeys: A model for spiral bacteria infection in humans ★

BACKGROUND/AIMS There is no generally accepted model for Helicobacter pylori infection in humans. The aim of this study was to examine the natural history and effect of treatment in rhesus monkeys and sequentially define the immune response to H. pylori in relation to treatment. METHODS Infection and gastritis were graded blindly by histological analysis and culture of biopsy specimens harvested during gastroduodenoscopies in 26 anesthetized colony-bred monkeys. Plasma H. pylori-specific immunoglobulin (Ig) G levels were determined by enzyme-linked immunosorbent assay. RESULTS H. pylori and Gastrospirilum hominis-like organisms were present in 13 and 9 monkeys, respectively; 3 animals harbored both organisms, whereas 4 monkeys were not infected. Gastritis score was < or = 1.5 in animals uninfected or infected only with G. hominis-like organisms and > or = 2.0 in all H. pylori-infected animals. IgG ratios were > or = 0.5 in 12 of 13 H. pylori-infected animals and in 2 of 13 H. pylori-negative animals (P < 0.001). One monkey became infected with H. pylori during the observation period, with concurrent increase of gastritis and plasma IgG levels. In untreated animals, infection, gastritis, and plasma IgG levels remained unchanged over 7-15 months. Triple therapy eradicated H. pylori at 6 months in 4 of 6 animals while suppressing gastritis and plasma IgG levels. CONCLUSIONS Rhesus monkeys harboring H. pylori are persistently infected and have gastritis and elevated specific IgG levels, all of which may respond to appropriate therapy, whereas G. hominis infection is associated with little inflammation.

Myoadenylate deaminase deficiency: Inherited and acquired forms☆

Myoadenylate deaminase deficiency, the most common of the known enzyme deficits of muscle, appears to occur in two forms. The primary type seems to be inherited as a complete gene block in an autosomal recessive pattern. Although occasionally diagnosed in infancy, when muscle biopsy is performed on a hypotonic but normoreflexic child, the deficiency is usually not symptomatic until adult or middle age, when muscle cramping and exercise intolerance develop. The skeletal muscle isozyme is immunologically, and presumably genetically, unique, and these patients have normal levels of adenylate deaminase in their other cells and tissues. A presumptive diagnosis can usually be made by an ischemic forearm exercise test, which shows a negligible increase in blood ammonia, despite a normal rise in lactate. Despite the absence of more than 99% of normal adenylate deaminase activity, the muscle biopsy shows no anatomic pathology, and other enzymes are at normal levels. These patients do not suffer progressive disease, and should be reassured, and encouraged to maintain physical activity. The heterozygous state is probably asymptomatic, except, perhaps, on extreme exercise, but may be associated with an increased incidence of malignant hyperthermia susceptibility. Since the gene defect is not rare, it is not surprising that some cases of the deficiency will be coincidentally associated with other neuromuscular disease. However, there is also a secondary form of myoadenylate deaminase deficiency, consequent to muscle damage from other disease. In this form, the residual activity is higher (1-10% of normal), may present rare foci of positive stain in the section, and reacts normally with antibody to the muscle isozyme. Other muscle enzymes are also depleted, although not as severely, and the prognosis in such cases is dictated by the primary disease. Since the heterozygous state is common, these patients might have been carriers, whose adenylate deaminase levels have been lowered for the deficient category by the advent of other neuromuscular disease.

Mutations in MCT1 cDNA in patients with symptomatic deficiency in lactate transport.

We identified 5 patients with subnormal erythrocyte lactate transport plus symptoms and signs of muscle injury on exercise and heat exposure. All had transport rates below the 95% envelope for normals. Three cases had rates 40–50% of mean normal. One was found to have a missense mutation in monocarboxylate transporter 1 (MCT1), the gene for the red cell lactate transporter (also expressed in skeletal muscle), at a conserved site, which was not mutated in a cohort of 90 normal humans. The other 2 cases had a different missense mutation (at a nonconserved site), which was also not mutated in the normal cohort. All 3 patients were heterozygotes. We presume that these mutations are responsible for their subnormal lactate transport, and hence their muscle injury under environmental stress; homozygous patients should be more seriously compromised. The other 2 cases had lactate transport rates 60–65% of mean normal, and their MCT1 revealed a third mutation, which proved to be a common polymorphism in the normal cohort. These 2 patients may be physiologic outliers in lactate transport, with their muscle damage arising from some other genetic defect.

Hydroxyurea: Mechanism of Action

Acetohydroxamic acid has been identified, by paper chromatography, in the blood of three patients with chronic myelogenous leukemia on hydroxyurea therapy. This suggests that the drug is hydrolyzed yielding hyroxylamine, which then cleaves thioesters, in particular acetyl-coenzyme A.

Preparation and some properties of stable and carbon-14 and tritium-labeled short-chain aliphatic hydroxamic acids☆

Abstract A simple and safe procedure has been described for the preparation of short-chain aliphatic hydroxamic acids in quantities as large as 1 mole and as small as 0.01 mole. The procedure is equally suitable for the preparation of isotopically labeled hydroxamates, as has been demonstrated in the case of 1-14C-acetohydroxamic acid and 3H-acetohydroxamic acid. Some physical and chemical characteristics, including infrared spectra of formo-, aceto-, propiono-, and isobutyro-hydroxamic acids prepared by this method have been described.

Relative distribution of three major lactate transporters in frozen human tissues and their localization in unfixed skeletal muscle.

We have prepared affinity‐purified rabbit polyclonal antibodies to the near‐C‐terminal peptides of human monocarboxylate transporters (MCTs) 1, 2, and 4 coupled to keyhole limpet hemocyanin. Each antiserum reacted only with its specific peptide antigen and gave a distinct molecular weight band (blocked by preincubation with antigen) after chemiluminescence reaction on Western blots from sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) of tissue membrane proteins. Densitometry showed distinctive expression patterns for each MCT in a panel of 15 frozen human tissues, with the distribution of MCT1 ≫L:MCT2>MCT4. Fluorescence microscopy of unfixed skeletal muscle using fluorescein‐conjugated secondary antibody was correlated with reverse adenosine triphosphatase (ATPase) stained sequential sections to identify fiber‐type localization. MCT1 expression was high in the sarcolemma of type 1 fibers, modest to low in type 2a fibers, and almost absent in type 2b fibers. In contrast, MCT4 expression was low to absent in the membrane of most type 1 fibers, but high in most 2a and in all 2b fibers, favoring the view that their high lactate levels during work may be channeled in part to neighboring type 1 (and perhaps 2a) fibers for oxidation, thereby delaying fatigue. MCT2 expression was limited to the sarcolemma of a type 1 fiber subset, which varied from <5 to 40%, depending on the specific muscle under study. Quantitative chemiluminescent densitometry of 10 muscle biopsies for their MCT2 and MCT4 content, each normalized to MCT1, confirmed the unique variation of MCT2 expression with biopsy site. The application of these antibodies should add to the understanding of motor unit physiology, and may contribute to the muscle‐biopsy assessment of low‐level denervation.

Expression of the monocarboxylate transporter MCT1 in the adult human brain cortex.

Distribution of the monocarboxylate transporter MCT1 has been investigated in the cortex of normal adult human brain. Similarly to the glucose transporter GLUT1 55 kDa isoform, MCT1 was found to be strongly expressed on blood vessels in all cortical layers. In addition, laminar analysis revealed intense MCT1 expression in the neuropil of layer IV in primary auditory (AI) and visual (VI) areas, while this expression was more homogeneous in the non-primary auditory area STA. The cellular distribution shows that MCT1 is strongly expressed by glial cells often associated with blood vessels that were identified as astrocytes. The observed distribution of MCT1 supports the concept that, under certain circumstances, monocarboxylates could be provided as energy substrates to the adult human brain. Moreover, the distinct laminar pattern of MCT1 expression between primary and non-primary cortical areas may reflect different types of neuronal activity requiring adequate supply of specific energy substrates.

Presence and localization of three lactic acid transporters (MCT1, -2, and -4) in separated human granulocytes, lymphocytes, and monocytes.

We fractionated leukocytes from three donors into >90% pure samples of granulocytes, lymphocytes, and monocytes and tested them for transcriptional and translational expression of three physiologically-proven lactate transporters, monocarboxylate transporter 1(MCT1), MCT2, and MCT4, using RT-PCR and affinity-purified rabbit antibody (Ab) to the C-terminal segment of each human MCT. Transcripts of all three MCTs were identified in each leukocyte fraction by RT-PCR and proven by sequencing of fragments extracted after isolation on agarose gels. Transporter protein of the appropriate size was demonstrated for each of the monocarboxylate transporters MCTs in lymphocytes and monocytes by Western blot, while lower-molecular-weight bands were found in granulocytes and are presumed to be degraded forms, because they were blocked by antibody-antigen (Ab-Ag) preincubation. IHC demonstrated all three MCTs in methanol-fixed droplets of all three leukocyte fractions; stain was abolished on omission of the primary Ab. Plasmalemmal staining occurred with all MCTs in all leukocyte fractions. Because the Km for lactate increases approximately fivefold at each step, with MCT2<1<4, leukocytes must use the full range of lactate binding to survive in acidic and hypoxic environments. Except for MCT4 in lymphocytes, all the MCTs also stained leukocyte cytoplasm, often with distinct granularity. Nuclear membrane staining was also seen with MCT1 and MCT2, while platelet plasmalemma stained only with MCT2.

Immunologic evidence for three isoforms of AMP deaminase (AMPD) in mature skeletal muscle

Four rabbit polyclonal antisera to purified AMP deaminase (AMPD) isozymes were used to precipitate homogenate AMPD activity from dissected gracilis, soleus and gastrocnemius muscles of the cat, rabbit, rat, mouse, Rhesus monkey, human and toad. The antisera were also tested against other unusual muscles: autonomically innervated striated muscle of the upper esophagus (UEM), skeletal muscle of patients with myo-AMPD deficiency and extraocular muscles (EOM) of humans and Rhesus monkeys. The reference antiserum, M, prepared against human psoas muscle AMPD, precipitated > 90% AMPD from all primate skeletal muscles tested, and from type-2 muscles of all mammals tested, but < 75% from cat and rodent soleus, toad gastrocnemius and primate UEM, EOM and myo-AMPD deficient muscles. Thus, a second isozyme was clearly indicated. Antibody B, against rat liver and kidney AMPD, had no effect with any muscle specimen. Antibody C, against rat heart AMPD, produced additive precipitation of AMPD from soleus of rat and mouse, while antibody E1, against human red cell (and heart) AMPD, produced additive AMPD precipitation from toad gastrocnemius, cat soleus and muscles of several AMPD-deficient humans. A second AMPD isozyme thus accounted for as much as 25% of total activity in some animal red muscles, but no more than 5% in human mixed muscles. At least one more isozyme is needed to account for muscle AMPD unreactive with all antibodies tested in rabbit soleus, toad gastrocnemius and primate UEM and EOM. A list is appended of the approximate AMPD activity in various human cells and tissues.